携带小鼠白介素-4截短型突变体基因的5型腺相关病毒载体的构建及外源蛋白的体外表达

Construction of Recombinant Adeno-associated Virus Encoding Interleukin-4 Antagonistic Mutant and Exogenous Protein Expression in vitro

目的:制备携带碳端结构域缺失的小鼠白介素-4(Interleukin-4,IL-4)基因突变体的5型重组腺相关病毒(recombinant adeno-associated virus, r AAV)并在细胞水平检测其介导的外源蛋白表达情况。方法:通过分子克隆技术构建携带小鼠IL-4碳端22个氨基酸缺失的突变体的表达质粒pSNAV-mIL-4ΔC22,三质粒共转染法制备重组的5型AAV病毒,体外感染人支气管上皮样细胞系16HBE和BEAS-2B,并通过Western blot和ELISA检测外源蛋白表达。结果:DNA测序表明构建的小鼠IL-4碳端第118位氨基酸位点处截短的突变体基因表达序列正确无误,制备的重组病毒载体r AAV5-mIL-4ΔC22滴度约为3×10^(11)vg/m L。r AAV5-GFP感染16HBE和BEAS-2B细胞后36小时开始可见持续稳定的荧光蛋白表达,重组病毒r AAV5-mIL-4ΔC22感染16HBE和BEAS-2B细胞后外源蛋白在培养上清中呈分泌型表达。结论:本研究成功构建了携带小鼠IL-4碳端结构域缺失型突变体的AAV表达质粒并制备了重组病毒r AAV5-mIL-4ΔC22,该病毒可有效转染16HBE和BEAS-2B细胞并介导外源基因分泌表达截短型小鼠IL-4突变体蛋白。

Objective: To prepare recombinant adeno-associated virus harboring truncated murine interleukin-4 mutant gene and detect the exogenous protein expression in vitro. Methods: An m IL-4 antagonistic mutant DNA expression plasmid pSNAV-mIL-4ΔC22 was constructed through gene clone technology, a recombinant virus vector r AAV5-mIL-4ΔC22 was prepared by three plasmids co-transfection, human bronchoid epithelioid cell line 16 HBE and BEAS-2 B were transfected with recombinant AAV5 vectors, then the expression of foreign protein was detected by Western blot and ELISA. Results: The mutant gene m IL-4ΔC22 was verified by DNA sequencing and the titer of virus vector r AAV5-mIL-4ΔC22 is about 3×1011vg/m L. 16 HBE and BEAS-2 B expressed GFP lastly and stably when 36 hours after receiving r AAV-GFP trensfection. Conclusions: Our study constructed recombinant vector r AAV5-mIL-4ΔC22 encoding murine IL-4 antagonistic mutant protein deleting the C-terminus region, the virus vector effectively transfected 16 HBE and BEAS-2 B, and mediated exogenous protein secretory expression in vitro.